Cell adhesive contacts are critical for the development and maintenance of multicellular organisms. These contacts are mediated by cell adhesion molecules (CAMs), a versatile class of compounds expressed on the cell surface. Cells adhere to one another and to extracellular substrates through the concerted action of a variety of CAMs, which act as both receptors and ligands on opposing cells. There are four subclasses of CAMs; selectins, cadherins, immunoglobulins and integrins. Selectins influence the localization of circulating leukocytes during inflammation, while cadherins and immunoglobulins establish and maintain cell-to-cell association and recognition mechanisms (Elangbam et al., Vet. Pathol., 1997, 34, 61-73). The fourth class of CAMs, known as integrins, play an important role in cell migration, cell anchorage to substrates and cytoadhesion signaling pathways (Akiyama, Hum. Cell, 1996, 9, 181-186).
Integrins are heterodimeric cation-dependent membrane glycoproteins composed of an alpha and beta subunit. To date, 8 beta and 15 alpha subunits have been identified and these subunits have been shown to combine to form over 20 different .alpha..beta. heterodimers. Integrins have been found in all tissues examined and consist of a large extracellular domain, a transmembrane domain and a smaller cytoplasmic domain. It is the extracellular domain of the integrin that acts as a receptor for various matrix proteins, while the cytoplasmic domain has been shown to interact with actin filaments of the cytoskeleton, thereby mediating signaling cascades (LaFlamme et al., Matrix Biol., 1997, 16, 153-163).
Integrin beta 3 (also known as human endothelial glycoprotein, GP3A, GPIIIa, ITGB3, CD61 and platelet glycoprotein 3a) is the common beta subunit partner of the members of the .beta.3 subfamily of integrins. This subfamily consists of two members, the vitronectin receptor, and the fibrinogen receptor and cells expressing this class of integrin receptor can adhere to various matrix proteins and participate in cytoadhesion driven cellular responses.
Integrin beta 3, in conjunction with integrin alpha v, forms the vitronectin receptor (.alpha.V.beta.3). This heterodimeric receptor is localized to platelets, endothelial cells, monocytes, macrophages and osteoclasts with the highest expression found in the osteoclasts (Rodan and Rodan, J. Endocrinol., 1997, 154 Suppl, S47-56). The vitronectin receptor functions to mediate the adhesion of cells to vitronectin, and a variety of extracellular matrix proteins. It is through a specific tripeptide sequence referred to as the RGD sequence, so named because of its amino acid composition (arginine-glycine-aspartic acid), that receptor-protein binding occurs. One category of inhibitors that targets the vitronectin receptor are peptidomimetics, designed to block the interactions between the receptor and RGD-containing proteins (Akiyama, Hum. Cell, 1996, 9, 181-186; Horton, Int. J. Biochem. Cell. Biol., 1997, 29, 721-725).
The activation of the vitronectin receptor has been shown to promote cellular migration and to provide signals in the regulation of cell proliferation and differentiation and to potentiate the effects of insulin (Ruoslahti, Kidney Int., 1997, 51, 1413-1417). Upregulation of the vitronectin receptor is associated with pathological conditions such as vascular restinosis (Clemetson and Clemetson, Cell. Mol. Life Sci., 1998, 54, 502-513), excessive bone resorption (Rodan and Rodan, J. Endocrinol., 1997, 154 Suppl, S47-56), and the process of angiogenesis during malignant melanomas (Cheresh, Cancer Metastasis Rev., 1991, 10, 3-10).
Integrin beta 3, in conjunction with integrin alpha IIb, also forms the fibrinogen receptor (.alpha.IIb/.beta.3) which mediates platelet aggregation. This receptor is basally inactive but can be activated by several agonists causing it to bind fibrinogen which then forms cross-bridges to fibrinogen receptors on adjacent cells. This receptor has also been shown to bind other proteins including fibronectin, von Willebrand factor (vWf) and vitronectin (Shattil, Thromb. Haemost., 1993, 70, 224-228).
Mutations in the gene of either subunit of the fibrinogen receptor, resulting in receptor deletion or misfunction, are the primary cause of the bleeding disorder known as Glanzman's thrombasthenia (GT) (Kato, Crit. Rev. Oncol. Hematol., 1997, 26, 1-23). This disorder is transmitted as an autosomal recessive trait and several mutations, including gene deletions, point mutations, and rearrangements have been identified (Kato, Crit. Rev. Oncol. Hematol., 1997, 26, 1-23).
Currently, therapeutic agents which affect the function of receptors containing the integrin beta 3 subunit have been designed to interfere with the binding properties of the receptor. As such, several inhibitors have been reported in the art and these include synthetic compounds and their derivatives, antibodies, and peptidomimetics, all of which act as antagonists to receptor ligand binding.
Abciximab, ReoPro.RTM. (Eli Lilly and Co.) is the Fab fragment of the chimeric human-murine monoclonal antibody 7E3. Abciximab binds to the glycoprotein (GP) IIb/IIIa (.alpha.IIb.beta.3) receptor of human platelets and inhibits platelet aggregation. The mechanism of action is thought to involve steric hindrance and/or conformational effects to block access of large molecules to the receptor rather than direct interaction with the RGD (arginine-glycine-aspartic acid) binding site of GPIIb/IIIa. Abciximab also binds with similar affinity to the vitronectin (.alpha.v.beta.3) receptor found on platelets and vessel wall endothelial and smooth muscle cells.
Abciximab is indicated as an adjunct to percutaneous coronary intervention for the prevention of cardiac ischemic complications. However, a potentially dangerous human antibody response to the chimeric antibody occurred in approximately 6% of patients (Clemetson and Clemetson, Cell. Mol. Life Sci., 1998, 54, 502-513).
Monoclonal antibodies to both integrin beta 3-containing receptors have been reported in the literature. Recently it was demonstrated that prostate carcinoma cells express the .alpha.IIb/.beta.3 fibrinogen receptor and that antibodies to this receptor were capable of inhibiting the invasive properties of the carcinomal cells (Trikha et al., Cancer Res., 1996, 56, 5071-5078).
Monoclonal antibodies produced by three different hybridoma cell lines that target the .alpha.v.beta.3 (vitronectin) receptor and inhibit the binding of fibronectin or vitronectin to osteoclasts are disclosed in U.S. Pat. Nos. 5,578,704, 5,652,109 and 5,652,110. These antibodies are used to detect the .alpha.V.beta.3 integrin and to treat disease conditions characterized by excessive bone resorption (Kim et al., 1997,; Kim et al., 1997,; Kim et al., 1996).
To date, no therapeutic agents that effectively inhibit the expression of integrin beta 3 have been identified. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting integrin beta 3 function by reducing the levels of expression.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of integrin beta 3 expression.